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Pyridinone or pyrimidinone nucleoside bases containing fused aromatic
polycyclic rings are provided. These polycyclic nucleosides are
incorporated into oligonucleotides and hybridized to complementary nucleic
acid. Fluorescence spectroscopy and thermal denaturation profiles provided
evidence that the polycyclic base is intercalated into the resulting
duplex. The fused polycyclic ring systems optionally are substituted with
reactive species which inactivate complementary nucleic acids. The
oligonucleotides of this invention are useful as improved probes,
diagnostic reagents, or for cleaving or derivatizing predetermined domains
within nucleic acids.

1. A pyridinone or pyrimidinone nucleoside comprising a fused aromatic polycyclic base having molecular dimensions equal to or less than about 30-50 angstroms by about 30-50 angstroms
by about 3-7 angstroms, wherein the nucleoside is represented by the structure ##STR18## wherein R.sub.3 is an aromatic polycycle comprising up to four fused aromatic rings;

13. An oligonucleotide of the structure: ##STR20## wherein A is an insoluble matrix or the nucleoside ##STR21## R.sub.5 is H or hydroxyl; R.sub.4 is O, S, alkyl, alkylamine, or alkyl ether; n is an integer; and B is a nucleoside base;
wherein at least one base is of the structure; ##STR22## and is positioned within about the first 20 percent or about the last 20 percent of the length of the oligonucleotide, and wherein

14. The oligonucleotide of claim 13 wherein the bases are guanidine, cytosine, adenosine, uracil, or thymine.

15. The oligonucleotide of claim 13 wherein n ranges about from 5 to 70.

16. The oligonucleotide of claim 13 wherein the polycyclic aromatic base is present in the second nucleoside 5' or 3' from the end of the oligonucleotide.

17. The oligonucleotide of claim 13 wherein the polycyclic aromatic base is fluorescent.

18. The oligonucleotide of claim 13 wherein the polycyclic aromatic base is ##STR23##

19. The compound of claim 13 wherein R.sub.3 is in substantially the same plane as the group ##STR24##

Description

This invention relates to nucleoside derivatives and oligonucleotides containing
such nucleosides. These derivatized nucleotides are capable of intercalating when incorporated into oligonucleotides and hybridized to complementary nucleic acids

It is known to employ oligonucleotides or oligonucleoside derivatives having an "anti-sense" sequence in attempts to inhibit the translation of mRNA having the complementary "sense" sequence. The anti-sense sequence hybridizes to the sense mRNA
in vivo, where it sterically blocks ribosomal translation of the mRNA (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83.4143 [1986]; Tullis, WO 83/01451, published 1983; and Goodchild et a1 , WO 87/07300, published 1987).

The phosphate backbone of the anti-sense oligonucleotides has been derivatized in an effort to stabilize the polymer and make it more cell permeable (Goodchild et al., op cit [triesters]; Marcus-Sekura et Nucl. Acids Res. 15(14):5749
[1987][methylphosphonate, alkyl phosphotriesters, and phosphorothioate]; Zon et al., U.S. Ser. No. 07/030,073, filed Mar. 25, 1987, now abandoned [phosphorothioate]; and Smith et al., Proc. Natl. Acad. Sci. USA 83:2787 [1986][methylphosphonates]). Unfortunately, the kinetics of anti-sense oligonucleotide interactions with sense mRNA in vivo are not favorable for optimal therapeutic effect if one is to rely solely on hybridization of complementary bases.

The enhanced thermal stability of oligonucleotides having tethered intercalating groups has created great interest in employing such deoxyoligonucleotides as anti-sense agents. In one example, it was shown that an intercalator linked to the
oligodeoxynucleotides could specifically inhibit the cytopathic effect of influenza virus in MDCK cells (Zerial et al., Nucl. Acids Res. 15:9909 [1987]) and the translation of specific mRNAs in cell-free systems (Toulme et al., Proc. Natl. Acad.
Sci. USA 83:1227 [1986] and Cazenave et al., Nucl. Acids Res. 15:4717 [1987]). The presence of the tethered intercalator is desirable for therapeutic or diagnostic applications since it increases the thermal stability of the mRNA-antisense DNA duplex
and reduces the rate of disassociation. However, an improved effect could be accomplished if the antisense oligomer was able to irreversibly inhibit translation. Attempts to accomplish this objective through cross. linking (Webb et al, J. Am. Chem.
Soc. 108:2764 [1986]; Iverson et al., J. Am. Chem. Soc. 109:1241 [1987]) or DNA cleaving reagents (Chu et al., Proc. Natl. Acad. Sci. USA 82:963 [1985]; Dreyer et al., Proc. Natl. Acad. Sci. USA 82:968 [1985]) have been reported.

Tethering an intercalating agent to an oligodeoxynucleotide with a flexible linker is undesirable because the tethered intercalating group is free to interact with biological systems or with substances other than the target complementary nucleic
acid or oligonucleotides, e.g. such cell systems as membranes and hydrophobic domains of cellular proteins such as receptors or enzymes. Similarly, tethered intercalating groups are free to avidly seek out hydrophobic surfaces in the diagnostic or
therapeutic environments, for example, polyolefin lab ware and the like, where nonspecific binding will interfere in hybridization assays or therapeutic delivery of the anti-sense oligonucleotide as the case may be.

Accordingly, it is an object to provide oligonucleotides containing intercalating bases which are sterically confined.

It is another object to provide nucleosides for use in preparing such oligonucleotides.

A still further object is to provide such improved oligonucleotides which are capable of hybridizing to the complementary nucleotide sequence, thereby translationally inactivating the targeted strand. This intercalating group is rigidly
confined, unlike the intercalating substituents of the art which are free to rotate and freely change their orientation.

These and other objects will be apparent from consideration of this invention as a whole.

SUMMARY OF THE INVENTION

We have determined that the objectives herein can be accomplished by fusing a substantially planar polycyclic aromatic ring system to a nucleoside base and incorporating such nucleotides into oligonucleotides. The derivatized oligonucleotides
hybridize to their complementary RNA or DNA strand, the modified nucleoside remaining unpaired but nonetheless intercalated between adjacent base pairs in the duplex in a precisely stereochemically defined manner. The resulting duplex is stabilized, but
since the intercalating group lacks the freedom to rotate or otherwise change its position with respect to the remainder of the molecule, other interactions aside from intercalation are minimized. The intercalating group is sterically confined by its
linkage to the sugar-phosphate backbone and by the presence of one or two flanking bases.

In light of the precise steric targeting made possible by this invention, the intercalating moiety is substituted with a reactive group capable of covalently modifying a predetermined site in the complementary domain. Such reactive groups
include cross-linking agents and phosphate bond cleaving agents. These reactive groups are sterically confined and less likely to interact with cellular components or nucleic acid at sites other than the target complementary sequence.

In an embodiment of the invention nucleoside derivatives that contain a substantially planar polycyclic fused base are synthesized by the novel reaction of (a) a pyridinone or pyrimidinone nucleoside substituted with a leaving group capable of
participating in a nucleophilic displacement reaction and (b) an aromatic diamine. The resulting polycyclic nucleoside is incorporated into an oligodeoxynucleotide by in vitro synthesis at a predetermined specific sequence position. It was found that
oligonucleotides containing an extra polycyclic base hybridize specifically to their complementary sequences and the resulting duplex shows enhanced thermal stability (depending on the context and nature of the base). These oligonucleotides therefore
are useful in diagnostic or therapeutic utilities which depend upon oligonucleotide hybridization.

The novel oligonucleotides are useful as hybridization probes. The fluorescence of the polycyclic base can be followed as an integral label and detected as a measure of the presence of a complementary nucleic acid. Alternatively, the
oligonucleotide is labelled by any other conventional method, e.g. by the use of a radioactive isotope of phosphorus. Nucleic acid detected by hybridization using the novel probes of this invention is diagnostically useful or may be employed in the
recombinant synthesis of polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a depicts fluorescence quenching upon hybridization of the oligonucleotides of this invention to their complementary strand. ##STR1##

FIG. 1b shows the melting curves for duplexes containing the oligonucleotides of this invention. ##STR2##

DETAILED DESCRIPTION OF THE INVENTION

The improved intercalating oligonucleotides of this invention have incorporated into their sequence at least one polycyclic base which is substantially planar. Substantially planar means that the steric bulk of the group lies substantially
within an envelope approximating the steric gap bounded by the sugar backbone and flanking bases present in a complementary nucleic acid strand. In general, this envelope has dimensions of about 30-50 Angstroms in width and depth and about 3.7 Angstroms
in thickness.

Typically, the polycyclic nucleoside will have the structure ##STR3## wherein R.sub.3 is an aromatic polycycle, Y is C or N, R.sub.7 is N or .dbd.C(R.sub.1)--, R.sub.1 and R.sub.6 are H or a radical and R.sub.2 is a ribose or deoxyribose sugar.

The key feature of this nucleoside is the presence of a fused aromatic polycycle. Since the polycycle is substantially unsaturated, it will not adopt the less planar profile of a saturated carbocycle. Further, since it is fused across the 3,4
or 1,6 positions, respectively, of the pyrimidinone or pyridinone residue, it is not free to rotate in relation to the same residue.

The structure of the aromatic polycycle is not critical so long as it is substantially hydrophobic and exhibits the desired molecular dimensions. Generally, it will contain about from 2 to 4 carbocyclic rings. However, it is also within the
scope herein that the aromatic polycycle contain a heterocyclic ring, usually a nitrogen or oxygen heterocycle, and including 5 or 6-membered rings containing one nitrogen or oxygen atom. Also included herein are aromatic polycycles containing rings
with substituents that do not substantially interrupt the above-described steric profile, e.g. hydroxyl, alkyl, hydroxyalkyl, carbonyl, ether or ester groups. Substituted aromatic polycycles of particular interest are quinones, especially
anthraquinones. When incorporated into an oligonucleotide, anthraquinone-substituted nucleosides are useful for hydrolyzing the phosphate backbone of complementary sequences under reducing conditions (NADH) in the presence of oxygen, thus irreversibly
inactivating the complementary strand. Similarly, other known reactive groups, e.g. cyanogen bromide activated methylthio, are substituted onto the aromatic polycycle. See, for example, Webb et al., Iverson et al., Chu et al., and Dreyer et al., all
cited above. Hybridization results in the cross-linking of the oligonucleotide to its complementary strand.

R.sub.1 and R.sub.6 are not critical substituents and in fact may be hydrogen or any radical, including halogen, ether, alkyl ether, saturated or unsaturated cycloalkyl, a heterocycle (N, S or O), hydroxyalkyl, ester, alkyl (e.g. C.sub.1 to
C.sub.10), or nitro. Ordinarily useful will be the substituents found at the analogous position in known pyrimidine nucleosides, including the naturally-occurring 4-amino pyrimidine nucleosides cytosine (R.sub.1 and R.sub.6 =H), 5-methylcytosine
(R.sub.1 =CH.sub.3, R.sub.6 =H), or 5-hydroxymethylcytosine (R.sub.1 =CH.sub.2 OH. R.sub.6 =H). Ordinarily R.sub.6 is hydrogen. For the purposes herein, neither R.sub.1 nor R.sub.6 are included within the calculation of the steric envelope described
above.

R.sub.2 is a deoxyribose or ribose sugar substituted at its anomeric carbon with the polycyclic base in either the .alpha. or .beta. configuration. Ordinarily R.sub.2 is a deoxyribose. The hydroxyl groups of R.sub.2 are, if necessary,
substituted with appropriate blocking groups such as dimethoxy trityl in order to protect the nucleoside for synthetic methods employed for incorporation of the nucleoside into an oligonucleotide.

The above described nucleosides are produced by reacting a polycyclic aromatic diamine with a pyrimidinone or pyridinone nucleoside substituted at the 4 or 6 positions, respectively, with a leaving group. Examples of suitable leaving groups are
##STR5## wherein R is alkyl or aryl, preferably para methyl phenyl or triisopropyl phenyl. The nucleoside starting materials are synthesized by known procedures See for example Bischofberger, Tet. Lett. 28:2821 (1987).

Nucleosides for which R.sub.7 =C can be produced by reacting an aromatic C-nucleophilic compound that has an amine or an amine precursor in an adjacent position with the same starting materials, e.g. o-Nitrophenylacetonitrile can be reacthed with
the 4-substituted pyrimidine nucleoside, followed by reduction of the nitro group to give an amine and subsequent cyclization to yield the polycyclic compound.

The aromatic diamine is any of the above-described aromatic polycycles substituted with at least two amino groups, preferably on adjacent ring system carbons. Alternatively, conducting the reaction with an amine alkyl substituted aromatic
polycycle will result in a fused ring containing 6 or more ring atoms. 1, 2,-diamino anthraquinone is commercially available. This is reacted with the leaving group-substituted nucleoside under basic conditions followed by acid catalyzed cyclisation in
the presence of dithionite to yield the polycyclic nucleoside.

The aromatic polycyclic nucleosides of this invention then are incorporated into oligonucleotides using known procedures. The resulting oligonucleotides have the structure ##STR6## wherein A is an insoluble matrix or the nucleoside ##STR7##
R.sub.5 is H or hydroxyl, R.sub.4 is O, S, alkyl, alkylamine or alkyl ether; n is an integer; and B is a base; provided, however, that at least one base is a substantially planar polycyclic base substantially having the dimensions of about 30-50
Angstroms x about 30-50 Angstroms x about 3-7 Angstroms.

The number of nucleotide bases, n, are those which are sufficient to produce an oligonucleotide that will hybridize to a complementary nucleic acid or oligonucleotide, n generally ranging about from 5 to 70. Typically, about 10 to 20
complementary bases will be sufficient to target the oligonucleotide to a unique sequence, but the number and identity of the bases will be determined by the artisan taking into account the hybridization conditions (stringent or physiologic) and the
degree of discrimination desired. There is no upper limit on n although little advantage is gained with more than 50 bases in light of the synthetic burden.

The polycyclic base will be present in the nucleoside at less than 10% of the total number of bases, and such bases should not be positioned adjacent to one another. Generally, only one such base will be present in the oligonucleotide and it is
best situated adjacent to either of the 5' or 3' terminal bases of the oligonucleotide. The remaining bases will be naturally occurring or, if not, they will be capable of base pairing with such natural bases.

The 5' end of the oligonucleotide may be linked to an insoluble support such as silica for the addition of more nucleosides by in vitro chemistries.

The nucleosides of this invention are useful in manufacturing the above described oligonucleotides for antiviral utilities. The oligonucleotides are useful as analytical probes. They are labelled using an appropriate radioisotope, e.g.
phosphorus 32, in the synthesis of the oligonucleotide, and the probe is used in the same way as oligonucleotide probes have been employed in the past. Certain of the polycyclic bases herein are fluorescent; this property may be assayed in order to
follow hybridization and avoids the use of radioisotopes or other exogenous labels. The intercalating capability of the oligonucleotides herein permits the use of more stringent conditions. e.g. higher washing temperatures, and hence will improve the
accuracy of analytical procedures and reduce background.

The reactive polycyclic substituents are analytically useful as well since it is straight-forward to follow their reaction in aqueous solution containing the target nucleic acid by electrophoretically separating the reaction products and
observing for the appearance of the reaction product. Where the reactive oligonucleotide contains a phosphate cleaving group, for example the anthraquinone, then one will identify two new bands representing the split target nucleic acid. On the other
hand, cross-linked nucleic acid will migrate in distinct fashion from that which has not undergone reaction.

The oligonucleotides of this invention also will have utility as agents for the anti-sense inhibition of translation of target nucleic acids. Such utilities have already been extensively explored with other anti-sense oligonucleotides and the
oligonucleotides herein will be used in substantially the same fashion.

The target nucleic acids include mRNA and DNA, and may be present in restriction enzyme digests or other fragments of nucleic acids, e.g. RFLPs. mRNA also is an appropriate target nucleic acid. Nucleic acid encoding any polypeptide is a
suitable target. Typically, nucleic acids encoding diagnostically or therapeutically meaningful proteins will be selected, for example viral proteins, oncogenes, growth factors, B-hemoglobin and the like. Further, other nucleic acids that encode no
protein may be of interest, e.g. transcription or translation control domains or sequences useful in forensic medicine.

In the inventive method of this invention, polycyclic nucleoside derivatives are synthesized by a novel, one pot procedure. In this method, a compound

(a) having the formula ##STR8## wherein L is a leaving group, R.sub.1 and R.sub.6 are H or a radical, Y is C or N and R.sub.2 is a ribose or deoxyribose sugar, is

(b) reacted with an aromatic polycyclic diamine or an aromatic C-nucleophile (e.g. cyano, convertible to the acid and decarboxylated to yield .dbd.CH--) having an amine or amine precursor in an adjacent position. For purposes herein, amine
includes an amine precursor.

The products of this novel reaction are polycyclic nucleoside derivatives. When incorporated into specific positions in oligonucleotides, they provide a model for studying intercalation related phenomena (Rebek et al., J. Am. Chem. Soc.
109:6866[1987]; Rebek et al., Ang. Chem. Int. Ed. Eng. 26:1244 [1987]; and Herbert et al., J. Med. Chem. 30:2081[1987]) as well as useful diagnostic or therapeutic reagents. Inspection of CPG-models of a double helical DNA containing an unpaired
naphth[2',3':4,5] imidazo[1,2-f]pyrimidine base reveals that the intercalated polycyclic base overlaps with the Watson-Crick hydrogen bonded cross section (Leonard et al., J. Am. Chem. Soc. 109:623[1987]). The geometry of the duplex closely resembles
an acridine intercalated into a DNA duplex. Moreover, compound 11 is fluorescent and consequently provides a convenient analytical handle for diagnostic hybridization assays. An important point in the incorporation of unnatural bases into
oligonucleotides by chemical synthesis on a solid support is the stability of the base to the conditions typically employed, i.e., oxidation (I.sub.2 /THF/H.sub.2 O) and deblocking (conc. NH.sub.4 OH/55.degree. C.). Both monomers 19 and 20 meet that
requirement. On the other hand, the highly fluorescent ethenoadenosine and ethenocytidine are known to be labile to I.sub.2 (Leonard, N.J. CRC Crit. Rev. Biochem. 15:125[1984] and Kusmierek et al., J. Org. Chem. 52:2374[1987]) and would present
difficulties for incorporation into oligodeoxynucleotides by chemical synthesis. The polycyclic base derived from 1,2-diaminoanthraquinone was not stable to the deblocking conditions, but deblocking could be accomplished by the usual ammonia treatment
in the presence of dithionite. This notwithstanding, an alternative method for the preparation of oligomers is to employ enzymatic catalysis, e.g. polynucleotide phosphorylase (Kusmierek et al. op cit).

The monomers 21 and 22 were incorporated (Example R) into oligodeoxynucleotide sequences at different positions and contexts. We investigated the influence of the additional base on the thermal stability of the duplexes. The positional effect
of an inserted naphth[2',3':4,5] imidazo[1,2-f] pyrimidine base was studied in the homooligomer duplex A.sub.14 /T.sub.14 and the results are given in Table I.

As can be seen, the highest stabilization resulted when the unnatural base was inserted between the two terminal base pairs as in the duplexes 5'-TXT.sub.13 /A.sub.14 and 5.dbd.-T.sub.13 XT/A.sub.14 (entries 2 and 8). Moving the extra base
further away from the terminal base pairs reduced the amount of stabilization and the presence of the base in the middle of the duplex as in T.sub.7 XT.sub.7 /A.sub.14 (entry 5) resulted in destabilization by 5.degree. C. This unexpected positional
effect of an inserted base finds two parallels in the literature. In one study, an acridine intercalator was attached through a linker arm of flexible length to various positions on an oligodeoxynucleotide and the greatest stabilization of the duplex
was found when the linker was attached to either the 5' or 3' end and had a length of five methylene units (Asseline et al., EMBO J. 3:795[1984]; Asseline et al., J. Biol. Chem. 260:8936[1985]; Asseline et al., Proc. Natl. Acad. Sci. USA
81:3297[1984]; Le Doan et al., Nucl. Acids Res. 19:7749[1987]; and Le Doan et al., Nucl. Acids Res. 15:8643 [1987]). This best fits a model where the acridine is intercalated in the duplex between the two terminal base pairs. Another report
relevant to our findings comes from an X-ray crystallographic study. Daunomycin was cocrystallized with the self-complementary oligodeoxynucleotide 5'-CGTACG and the daunomycin was found to be intercalated in the terminal CG base pairs (Quigley et al.,
Proc. Natl. Acad. Sci. USA 77:7204[1980]).

These results can be rationalized by taking into account the different energetic contributions of the extra base to the stability of the duplex. The presence of the additional base stabilizes the duplex due to hydrophobic and base stacking
interactions. On the other hand, it causes geometric distortions to the helical structure which destabilizes the duplex. The amount of this unfavorable contribution to the duplex stability is dependent on the position of the inserted base. Being
positioned close to the end, it causes less long-range distortions than in the middle. Accordingly, a polycyclic base typically is positioned within about the first 20 percent or about the last 20 percent of the length of the oligonucleotide, and
ordinarily between the two 5' or 3' terminal bases.

The sequence specificity of the inserted unnatural base was studied and the results are shown in Table II.

Duplexes containing an extra unpaired base have been studied by .sup.1 H NMR spectroscopy and X-ray crystallography as model systems for frameshift mutagenesis (Patel et al., Biochemistry 21:445[1982]; Patel et al., Biochemistry 21:451[1982]; and
Saper et al., J. Mol. Biol. 188:111[1986]). NMR studies have shown that a duplex containing an extra unpaired deoxyadenosine exists with the adenine being stacked into the duplex (Hare et al., Biochemistry 25:7456 [1986]). Thermal denaturation studies
of these duplexes bearing the extra deoxyadenosine demonstrated that the presence of the extra base has a destabilizing effect on the duplex (Patel et al., supra and Saper et al., supra). It can be seen that the presence of an extra adenosine reduces
the stability of the duplex by approximately 2.5.degree. C., consistent with other findings (Zerial et al, supra). Insertion of the tetracyclic bases, on the other hand, causes a stabilization of the duplex, the pyrimido[1,6'-a]perimidine showing a
higher stabilization. Clearly, context effects are present but difficult to rationalize without knowledge of the detailed geometry.

The duplexes containing two inserted bases show a peculiar phenomenon (entries 6-9, Table II). With the extra base present in opposite strands (entries 6 and 7), the duplex shows a cooperative melting curve and the amount of duplex stabilization
is approximately the sum of the stabilization caused by both single insertions. When the two extra nucleotides are incorporated into the same single oligomer, however, the two inserted bases display anticooperative melting behavior and no increase in
T.sub.m is observed (entry 8) or a biphasic melting curve is obtained (entry 9, FIG. 1b). Again, these results cannot be rationalized without detailed structural information but they are suggestive of the existence of long-range changes in the helical
structure caused by the presence of the extra tetracyclic base.

In an attempt to synthesize the tricyclic nucleoside derivative 3, the 4-O-TPS pyrimidine derivative 1 (Bischofberger, N. Tet. Lett. 28:2821[1987]) was treated with o-phenylenediamine in refluxing THF and a slower moving spot was observed on
TLC, corresponding presumably to the adduct 2 (see below). The reaction did not stop at that stage, however, and a new fluorescent product of higher R.sub.f formed and was isolated in 49% yield. Quite unexpectedly this product still contained Br but
had lost NH.sub.3 according to MS and elemental analyses. The .lambda..sub.max in the UV spectrum was shifted to longer wavelengths relative to the starting o-phenylenediamine (.lambda..sub.max =294 nm) and exhibited a well resolved vibrational
structure. On the basis of these data, the product was subsequently assigned structure 4. This assignment was also confirmed by the .sup.1 H NMR spectrum in which the signal of the H-C9 is shifted to high-field due to the anisotropy of the neighboring
carbonyl group (Gunther, H. NMR-Spektroskopie; Georg Thieme Verlag; Stuttgart, 1973; pp 75-82). ##STR12##

In order to examine the generality of this rather unusual reaction the 4-O-TPS-thymidine and 4-O-TPS-deoxyuridine derivatives 5, 6 and 7 were reacted with various aromatic diamines and the cyclized products 8 (from o-phenylenediamine), 10 (from
1,8-diaminonaphthalene), 11 and 12 (from 2,3-diaminonaphthalene) and the isomeric compounds 13 and 14 (from 4-nitro-o-phenylenediamine were isolated in modest to good yields in modest to good yields (see infra). ##STR13##

When the reaction was carried out in the presence of base none of the cyclized compounds were obtained but the reaction stopped at the intermediate stage, e.g., treatment of 6 with 2,3-diaminonaphthalene in the presence of 1 equivalent of
ethyldiisopropylamine yielded 15 in 60% yield. 15 was slowly converted to 11 in refluxing THF, more rapidly in the presence of acetic acid demonstrating that this second cyclization step is acid catalyzed.

Alternatively, the reaction was carried out in two steps by first treating the 4-O-TPS derivative with the diamine in the presence of base and then affecting the cyclization with acid. By treating o-phenylenediamine and 7 with potassium
hexamethyldisilazane in THF at -78.degree. C., work up and subsequent reflux of the crude product in THF in the presence of 0.8 equivalents of acetic acid the cyclized product 9 was obtained in 60% overall yield. Similarly, by treating 7 and
1,2-diaminoanthraquinone with potassium hexamethyldisilazane in THF, work-up and subsequent treatment of the crude mixture with tetrabutyl ammonium fluoride followed by heating in acetic acid in the presence of dithionite the cyclized polycycle 23 was
obtained. ##STR14##

On the other hand, reaction of 7 with ethylene diamine and of 6 with 2,3-diaminopyridine led only to the adducts 16 and 17, respectively, which could not be cyclized under a variety of reaction conditions. ##STR15##

Also attempts to extend this reaction to purine nucleosides failed. The reaction of o-phenylenediamine with N.sup.2, 3 ,5'-triisobutyryl-6-O-TPS-dG resulted only in the formation of 18 which did not undergo any cyclization.

Without being limited to any particular theory of operation, a possible mechanism of this novel cyclization is shown below. ##STR16## It might involve initial Michael addition of the primary amine in a to form b, followed by ring opening
(b>c) and cyclization with los of NH.sub.3 to give the cyclized product (c>d).

10 and 11 on treatment with conc. NH.sub.3 rapidly gave the deacetylated derivatives which proved to be stable to further ammonia treatment at 55.degree. C. as observed on TLC and were directly dimethoxytritylated (DMT-C1/pyridine) yielding 19
and 20 and subsequently converted to the phosphonate triethylammonium salts 21 and 22. 21 and 22 were then incorporated into oligonucleotides using the standard H-phosphonate protocol (Froehler et al., Tet. Lett. 27:469[1986]and Froehler et al., Nucl. Acids. Res. 14:5399 [1986]). ##STR17##

The oligomers obtained after deblocking were purified by gel electrophoresis and desalted by reversed phase chromatography using a C.sub.18 Sep pack column. The presence of the tetracyclic bases in the oligomers was confirmed by UV and
fluorescence spectroscopies. The amount of fluorescence quenching in the single stranded oligomers containing the fluorescent naphth[2',3':4,5]imidazo[1,2-f]pyrimidine ring system was determined by snake venom phosphodiesterase digest and was found to
be approximately 50%.

EXAMPLES

2'-Deoxyuridine and thymidine were purchased from Sigma Chemical Co.; all other reagents were purchased from Aldrich Chemical Co. .sup.1 H NMR spectra were obtained using an 80 MHz IBM NR/80 spectrometer and recorded as ppm (.delta.) using TMS
as an internal standard. Mass spectra were obtained using the positive-ion fast-atom-bombardment (FAB) technique on a Hewlett-Packard 5985C instrument. UV spectra were obtained on a Beckman UV-7 spectrophotometer using methanol as the solvent.
Fluorescence spectra were obtained using an SLM 8000C spectrofluorometer. For thin layer chromatography (TLC) EM DC-Alufolien Kieselgel-60 F.sub.254 plates were used; column chromatography was performed with EM Kieselgel-60 (70-230 mesh). Melting
points were determined on a Buchi 510 capillary melting point apparatus and are uncorrected. Elemental analyses were done by Chemical Analytical Services, University of California, Berkeley, Calif. Oligonucleotides were synthesized on a Biosearch Model
4000 DNA synthesizer.

Oligonucleotide Synthesis. Polymer bound nucleotide H-phosphonates were prepared as previously described (Froehler et al., Tet. Let. 27:469[1986]) on control pore glass using the DBU salts of the protected nucleoside H-phosphonates. For
introducing the polycyclic nucleosides, a solution of .sup..about. 25 mg of the triethylammonium salts 21 and 22, respectively in 1 mL pyridine/acetonitrile 1:1 was used in the automated synthesis. For efficient coupling the wait time in the programmed
coupling step for 21 and 22 was increased from 6.times.8 sec to 26.times.8 sec. After oxidation and deblocking, the fragments were purified by polyacrylamide gel electrophoresis and the fluorescent (in the case of 17) or yellow (in the case of 19) bands
were eluted and the eluate desalted by loading onto a reversed phase C.sub.18 SepPac column (Waters Associates), washing with H.sub.2 O and finally eluting the fragments with 25% aqueous acetonitrile.

EXAMPLE S

Fluorescence Studies. Addition of the complementary undecamer 5'-ACACATCACTG to the fluorescent 5'-CAG TGA TGT GXT dodecamer caused a decrease in the fluorescence intensity whereas addition of random DNA sequences to the fluorescent dodecamer
had no effect, proving that the fluorescence intensity decrease is specific to the complementary oligomer. Also the fluorescence intensity did not further change after one equivalent of the complementary strand had been added, thus indicating the
formation of a duplex. The fluorescence intensity could be increased to the original level on heating as shown in FIG. 1a, consistent with the thermal denaturation of the duplex. The other dodecamers containing the
naphth[2',3':4,5]imidazo[1,2f]pyrimidine base gave similar results, with varying levels of fluorescence quenching. Other bases within the scope herein are useful in similar fashion for the assay of hybridization, which as shown is readily followed by
changes in fluorescence of the oligonucleotide. Such changes may include changes in fluorescence intensity (quenching or enhancement), wavelength (emission or adsorption), and the time course of fluorescence (energy transfer).

EXAMPLE T

Thermal Denaturation Profiles. The melting curves of the duplexes containing one or two of the tetracyclic bases derived from monomers 21 and 22, respectively, were measured at 2 mM oligomer concentration in buffered solution (100 mM NaCl, 10 mM
N.sub.a2 HPO.sub.4, 1 mM EDTA, pH=7.5). For comparison and reference, the melting profiles of the corresponding duplexes containing no extra bases and of the duplexes containing an extra adenosine were also measured. From these melting curves, the
T.sub.m of the duplexes were determined and the results are depicted in Tables I and II and FIG. 1b.

EXAMPLE U

Hypochromicity measurements. Hypochromicities of the duplexes were measured at 260 nm with a Kontron Uvikon 810 spectrophotometer in a 1 cm masked cuvette. The samples were 100 mM NaCl, 10 mM Na.sub.2 HPO.sub.4, 1 mM EDTA at pH 7.2 and
contained the oligomers at a concentration of 2 .mu.M in a total volume of 1 mL. The extinction coefficients (.xi.) of the oligomers were calculated and the .xi. of the oligomers containing the excra tetracyclic bases were approximated by adding the
values of the .xi. of the monomers at 260 nm (for 10:.xi. (260)=15 mL/.mu.mol; for 11:.xi.(260)=6 mL/.mu.mol) to the .xi. of the oligomers devoid of the tetracyclic bases. The samples were degassed with He, heated to .sup.- 55.degree. C. for 4 h,
allowed to cool and maintained at 0.degree. C. overnight. The absorbance of the samples was monitored from 15.degree. C. to .sup..about. 80.degree. C., increasing the temperature at a rate of 0.2.degree. C./min. Each sample was measured at least
twice and the data were separately normalized to % denaturation (% denaturation=100% [A.sub.o -A.sub.i ]/[A.sub.f -A.sub.i ], where A.sub.o = observed abs, A.sub.i =initial abs, A.sub.f =final abs.) and combined to obtain a melting curve. A linear least
squares analysis of this data gave a slope of transition and y-intercept from which the T.sub.m values were calculated.